Melanocyte stem cells (McSCs) sustain a healthy population of melanocytes throughout life. Understanding their behavior and regulation is relevant to disorders resulting from melanocyte loss such as post inflammatory hypopigmentation, vitiligo, and hair graying, as well as to the biology of melanoma. Currently, little is known about the cellular and molecular mechanisms that govern McSCs. The goal of this grant is to understand the function of Wnt signaling and its regulation in McSCs residing in the hair follicle. McSCs are coordinately activated with follicular epithelial stem cells (EpSCs) during the cyclical regeneration of the hair follicle. They provide differentiated melanocytes to the hair bulb for the production of hair pigment. We discovered that McSCs activate Wnt signaling following Wnt ligand secretion from adjacent EpSCs at the onset of anagen. By transgenically deleting or activating 2-catenin, a key mediator of Wnt signaling in McSCs, we have shown that Wnt signaling is crucial for McSC differentiation. We hypothesize that Wnt signaling is required for McSC differentiation and that EpSCs coordinately regulate Wnt signaling in McSCs and EpSCs themselves during the hair follicle cycle. To address this hypothesis, we will pursue the following specific aims. In Aim1, we will inhibit Wnt signaling in McSCs by expressing either a secreted inhibitor of Wnt signaling, Dkk1, or a dominant- negative form of a nuclear mediator of Wnt signaling, Lef1, in melanocytes. We will examine how this influences their ability to give rise to functional melanocytes during the hair cycle. In Aim2, to determine if Wnt ligand(s) secreted by EpSCs can activate Wnt signaling in neighboring McSCs and promote their differentiation, we will delete the Wntless gene, which is necessary for Wnt ligand secretion, in follicular EpSCs or force expression of a Wnt ligand by EpSCs, using genetic mouse models. In Aim3, we will test if Wnt inhibitors can promote McSC maintenance during aging, a process known to result in McSC exhaustion and loss following their aberrant differentiation. Specifically, we will utilize transgenic mouse models to overexpress either Dkk1 or a dominant-negative form of Lef1 in McSCs to examine if this promotes the maintenance of undifferentiated McSCs during aging. We will also test if Wnt inhibition can reverse McSC differentiation, a process that presages their ensuing loss. Our study will provide us with a novel understanding of the molecular mechanisms regulating McSCs and how melanocyte behavior is coordinated with hair regeneration, providing a scientific foundation for understanding diseases in which melanocytes are either inappropriately lost such as hair graying and vitiligo or undergo uncontrolled cell growth as in melanoma.